Bi-allelic genetic variants in the translational GTPases GTPBP1 and GTPBP2 cause a distinct identical neurodevelopmental syndrome.

The homologous genes GTPBP1 and GTPBP2 encode GTP-binding proteins 1 and 2, which are involved in ribosomal homeostasis. Pathogenic variants in GTPBP2 were recently shown to be an ultra-rare cause of neurodegenerative or neurodevelopmental disorders (NDDs). Until now, no human phenotype has been linked to GTPBP1. Here, we describe individuals carrying bi-allelic GTPBP1 variants that display an identical phenotype with GTPBP2 and characterize the overall spectrum of GTP-binding protein (1/2)-related disorders. In this study, 20 individuals from 16 families with distinct NDDs and syndromic facial features were investigated by whole-exome (WES) or whole-genome (WGS) sequencing. To assess the functional impact of the identified genetic variants, semi-quantitative PCR, western blot, and ribosome profiling assays were performed in fibroblasts from affected individuals. We also investigated the effect of reducing expression of CG2017, an ortholog of human GTPBP1/2, in the fruit fly Drosophila melanogaster. Individuals with bi-allelic GTPBP1 or GTPBP2 variants presented with microcephaly, profound neurodevelopmental impairment, pathognomonic craniofacial features, and ectodermal defects. Abnormal vision and/or hearing, progressive spasticity, choreoathetoid movements, refractory epilepsy, and brain atrophy were part of the core phenotype of this syndrome. Cell line studies identified a loss-of-function (LoF) impact of the disease-associated variants but no significant abnormalities on ribosome profiling. Reduced expression of CG2017 isoforms was associated with locomotor impairment in Drosophila. In conclusion, bi-allelic GTPBP1 and GTPBP2 LoF variants cause an identical, distinct neurodevelopmental syndrome. Mutant CG2017 knockout flies display motor impairment, highlighting the conserved role for GTP-binding proteins in CNS development across species.

RAF1 gene fusions are recurrent driver events in infantile fibrosarcoma-like mesenchymal tumors.

Infantile fibrosarcomas (IFS) and congenital mesoblastic nephroma (CMN) are rare myofibroblastic tumors of infancy and early childhood commonly harboring the ETV6::NTRK3 gene fusion. IFS/CMN are considered as tumors with an 'intermediate prognosis' as they are locally aggressive, but rarely metastasize, and generally have a favorable outcome. A fraction of IFS/CMN-related neoplasms are negative for the ETV6::NTRK3 gene rearrangement and are characterized by other chimeric proteins promoting MAPK signaling upregulation. In a large proportion of these tumors, which are classified as IFS-like mesenchymal neoplasms, the contributing molecular events remain to be identified. Here, we report three distinct rearrangements involving RAF1 among eight ETV6::NTRK3 gene fusion-negative tumors with an original histological diagnosis of IFS/CMN. The three fusion proteins retain the entire catalytic domain of the kinase. Two chimeric products, GOLGA4::RAF1 and LRRFIP2::RAF1, had previously been reported as driver events in different cancers, whereas the third, CLIP1::RAF1, represents a novel fusion protein. We demonstrate that CLIP1::RAF1 acts as a bona fide oncoprotein promoting cell proliferation and migration through constitutive upregulation of MAPK signaling. We show that the CLIP1::RAF1 hyperactive behavior does not require RAS activation and is mediated by constitutive 14-3-3 protein-independent dimerization of the chimeric protein. As previously reported for the ETV6::NTRK3 fusion protein, CLIP1::RAF1 similarly upregulates PI3K-AKT signaling. Our findings document that RAF1 gene rearrangements represent a recurrent event in ETV6::NTRK3-negative IFS/CMN and provide a rationale for the use of inhibitors directed to suppress MAPK and PI3K-AKT signaling in these cancers. © 2024 The Pathological Society of Great Britain and Ireland.

Spindle Cell Lesions with Oncogenic EGFR Kinase Domain Aberrations: Expanding the Spectrum of Protein Kinase-Related Mesenchymal Tumors.

EGFR aberrations are reported in a subset of myofibroblastic lesions with kinase domain duplication (EGFR-KDD) and exon 20 mutations being assigned to infantile fibrosarcomas (IFS), mesoblastic nephroma, and fibrous hamartoma of infancy (FHI), respectively. In this retrospective study, we correlated molecular findings with the histomorphology of 14 myofibroblastic lesions harboring such genetic changes identified by NGS. We additionally performed DNA methylation profiling (DNAmp) and immunohistochemistry. Lesions were from 10 males and 4 females with a mean age of 3 years (range, 0.3-14) and occurred subcutaneously in the upper limbs (n = 5), lower limbs (n = 3), back/thorax (n = 5), and the nasal cavity (n = 1). Eleven were cured by surgery, including 1 relapsed case. Two patients were lost to follow-up. One case was very recent, and the patient was biopsied. Histologically, the lesions showed a wide spectrum varying from classic FHI (n = 9) to IFS (n = 1) or lipofibromatosis-like tumors (LFT-like) (n = 2) or dermatofibrosarcoma protuberans-like (DFSP-like) (n = 1) to a predominantly myxoid spindle cell lesion (n = 1). Immunohistochemically, all neoplasms stained with CD34, whereas S100 was positive in 2/14. EGFR expression was observed in 9/10 cases. Molecularly, the IFS and 1 LFT-like harbored EGFR-KDD, whereas an exon 20 mutation was identified in all FHI, 1 LFT-like, the DFSP-like, and in predominant myxoid spindle cell lesion. By DNAmp, all but 2 cases formed a well-defined cluster, demonstrating that these lesions are also epigenetically related. In conclusion, EGFR kinase domain aberrations found in FHI, IFS, LFT-like, DFSP-like, and a spindle cell lesion with a predominant myxoid stroma of children and adolescents showed that these neoplasms with a broad morphologic spectrum belong to the group of protein kinase-related lesions with a distinct epigenetic signature. Molecular analyses, including DNAmp, help to identify and characterize this emerging category and become mandatory when targeted treatment is considered.

PATZ1-Rearranged Tumors of the Central Nervous System: Characterization of a Pediatric Series of Seven Cases.

PATZ1-rearranged sarcomas are well-recognized tumors as part of the family of round cell sarcoma with EWSR1-non-ETS fusions. Whether PATZ1-rearranged central nervous system (CNS) tumors are a distinct tumor type is debatable. We thoroughly characterized a pediatric series of PATZ1-rearranged CNS tumors by chromosome microarray analysis (CMA), DNA methylation analysis, gene expression profiling and, when frozen tissue is available, optical genome mapping (OGM). The series consisted of 7 cases (M:F=1.3:1, 1-17 years, median 12). On MRI, the tumors were supratentorial in close relation to the lateral ventricles (intraventricular or iuxtaventricular), preferentially located in the occipital lobe. Two major histologic groups were identified: one (4 cases) with an overall glial appearance, indicated as "neuroepithelial" (NET) by analogy with the corresponding methylation class (MC); the other (3 cases) with a predominant spindle cell sarcoma morphology, indicated as "sarcomatous" (SM). A single distinct methylation cluster encompassing both groups was identified by multidimensional scaling analysis. Despite the epigenetic homogeneity, unsupervised clustering analysis of gene expression profiles revealed 2 distinct transcriptional subgroups correlating with the histologic phenotypes. Interestingly, genes implicated in epithelial-mesenchymal transition and extracellular matrix composition were enriched in the subgroup associated to the SM phenotype. The combined use of CMA and OGM enabled the identification of chromosome 22 chromothripsis in all cases suitable for the analyses, explaining the physical association of PATZ1 to EWSR1 or MN1. Six patients are currently disease-free (median follow-up 30 months, range 12-92). One patient of the SM group developed spinal metastases at 26 months from diagnosis and is currently receiving multimodal therapy (42 months). Our data suggest that PATZ1-CNS tumors are defined by chromosome 22 chromothripsis as causative of PATZ1 fusion, show peculiar MRI features (eg, relation to lateral ventricles, supratentorial frequently posterior site), and, although epigenetically homogenous, encompass 2 distinct histologic and transcriptional subgroups.

A Novel NUTM1-NSMCE2 Fusion Gene in a Pediatric Chest NUT Carcinoma.

Nuclear protein of the testis carcinoma is an exceedingly rare and poorly differentiated carcinoma characterized by BDR4::NUTM1 gene translocation. Typically, the tumor affects young adults, and no standardized recommendations for therapeutic management have been available since 2022; the clinical course remains mostly dismal. We report the successful multimodal treatment of a 13-year-old boy affected by a primary chest NUT-carcinoma with a novel NUTM1 rearrangement that remains in complete continuous remission at 30 months from diagnosis.

LMNA::NTRK1 and PRDX1::NTRK1 Atypical Spitz Tumor: A Report of Two Additional Cases With Histological, Immunohistochemical, and Molecular Insights.

ABSTRACT: Over the past decade, advancements in molecular biology have contributed to changes in the diagnostic classification of Spitz neoplasms, including Spitz nevi, atypical Spitz tumors, and Spitz melanomas. The recent World Health Organization classification of skin tumors identifies fusion kinases, including NTRK1, NTRK2, and NTRK3, as critical drivers of these lesions. New fusion genes have continued to expand the spectrum of known molecular alterations, particularly within the category of Spitz NTRK-rearranged lesions. We present 2 new cases of NTRK-rearranged Spitz lesions: an atypical Spitz tumor with common LMNA::NTRK1 fusion and an atypical Spitz tumor with a rare PRDX1::NTRK1 fusion. Clinical, histopathological, immunohistochemical, and molecular analyses were performed to diagnose these patients. This report adds to the growing body of knowledge on NTRK-rearranged Spitz lesions and underscores the importance of integrating molecular findings with morphological and immunohistochemical data for the accurate classification and understanding of these neoplasms.

Bi-allelic LoF NRROS Variants Impairing Active TGF-ß1 Delivery Cause a Severe Infantile-Onset Neurodegenerative Condition with Intracranial Calcification.

Negative regulator of reactive oxygen species (NRROS) is a leucine-rich repeat-containing protein that uniquely associates with latent transforming growth factor beta-1 (TGF- ß1) and anchors it on the cell surface; this anchoring is required for activation of TGF-ß1 in macrophages and microglia. We report six individuals from four families with bi-allelic variants in NRROS. All affected individuals had neurodegenerative disease with refractory epilepsy, developmental regression, and reduced white matter volume with delayed myelination. The clinical course in affected individuals began with normal development or mild developmental delay, and the onset of seizures occurred within the first year of life, followed by developmental regression. Intracranial calcification was detected in three individuals. The phenotypic features in affected individuals are consistent with those observed in the Nrros knockout mouse, and they overlap with those seen in the human condition associated with TGF-ß1 deficiency. The disease-causing NRROS variants involve two significant functional NRROS domains. These variants result in aberrant NRROS proteins with impaired ability to anchor latent TGF-ß1 on the cell surface. Using confocal microscopy in HEK293T cells, we demonstrate that wild-type and mutant NRROS proteins co-localize with latent TGF-ß1 intracellularly. However, using flow cytometry, we show that our mutant NRROS proteins fail to anchor latent TGF-ß1 at the cell surface in comparison to wild-type NRROS. Moreover, wild-type NRROS rescues the defect of our disease-associated mutants in presenting latent TGF-ß1 to the cell surface. Taken together, our findings suggest that loss of NRROS function causes a severe childhood-onset neurodegenerative condition with features suggestive of a disordered response to inflammation.

CAPRIN1P512L causes aberrant protein aggregation and associates with early-onset ataxia.

CAPRIN1 is a ubiquitously expressed protein, abundant in the brain, where it regulates the transport and translation of mRNAs of genes involved in synaptic plasticity. Here we describe two unrelated children, who developed early-onset ataxia, dysarthria, cognitive decline and muscle weakness. Trio exome sequencing unraveled the identical de novo c.1535C > T (p.Pro512Leu) missense variant in CAPRIN1, affecting a highly conserved residue. In silico analyses predict an increased aggregation propensity of the mutated protein. Indeed, overexpressed CAPRIN1P512L forms insoluble ubiquitinated aggregates, sequestrating proteins associated with neurodegenerative disorders (ATXN2, GEMIN5, SNRNP200 and SNCA). Moreover, the CAPRIN1P512L mutation in isogenic iPSC-derived cortical neurons causes reduced neuronal activity and altered stress granule dynamics. Furthermore, nano-differential scanning fluorimetry reveals that CAPRIN1P512L aggregation is strongly enhanced by RNA in vitro. These findings associate the gain-of-function Pro512Leu mutation to early-onset ataxia and neurodegeneration, unveiling a critical residue of CAPRIN1 and a key role of RNA-protein interactions.

Paediatric astroblastoma-like neuroepithelial tumour of the spinal cord with a MAMLD1-BEND2 rearrangement.

Astroblastomas are neuroepithelial tumours defined by the presence of MN1 rearrangement and are typically located in the cerebral hemispheres. Rare cases of astroblastoma-like tumours carrying an EWSR1-BEND2 fusion have been recently described in the brain stem and spinal cord. We report a paediatric case of neuroepithelial astroblastoma-like tumour occurring in the spine and carrying a novel MAMLD1-BEND2 fusion. We believe that our case aligns with the rare astroblastoma-like tumours with EWSR1-BEND2 fusion, in terms of non-hemispheric location, pathology, methylation profile and activation of BEND2 transcription. Whether they may represent a distinct entity or a variant of MN1-altered astroblastoma is not clear.

Angioimmunoblastic T-Cell Lymphoma with Exuberant CD30-Positive Follicular Dendritic Cell Proliferation in a SARS-CoV-2 Patient: The Role of Mutational Analysis to Exclude an Associated Follicular Dendritic Cell Sarcoma.

Follicular dendritic cell (FDC) proliferation in angioimmunoblastic T-cell lymphoma (AITL) is still not well defined, challenging the accurate differential diagnosis between the AITL with expanded follicular dendritic cell meshwork and the combined AITL and follicular dendritic cell sarcoma (FDCS). Herein, we reported the case of a 58-year-old male with coexisting SARS-CoV-2 infection and AITL with an exuberant CD30-positive FDC proliferation, in which genetic analysis identified mutations of genes commonly involved in AITL but not in FDC sarcoma (i.e., RHOA, TET2, DNMT3A, and IDH2), thus supporting the reactive nature of the CD30-positive FDC expansion.

A novel BRD4-LEUTX fusion in a pediatric sarcoma with epithelioid morphology and diffuse S100 expression.

Malignant epithelioid soft tissue tumors encompass a wide spectrum of lesions. Among them, Epithelioid Malignant Peripheral Nerve Sheath Tumors (MPNST) constitute a distinct subgroup, accounting for <5% of all MPNST. Epithelioid MPNST are infrequently associated with neurofibromatosis type 1, occasionally arise in a schwannoma and show diffuse S100 and CD34 expression, often combined with INI-1 loss. However, the molecular mechanisms underlying the tumorigenesis of epithelioid MPNST remain largely unknown. We describe a case of a 10-year-old girl with an epithelioid malignancy of the orbit. The tumor proved positive for S100, CD34 and SOX10, and, although INI-1 expression was maintained, the overall features suggested the possibility of an epithelioid MPNST, arising in an unusual location. NGS analysis revealed a novel in-frame BRD4-LEUTX fusion gene. LEUTX plays an important role in embryonal genome activation and its expression is mostly suppressed postnatally. We were able to detect increased levels of LEUTX transcript in the tumor, indicating that BRD4-LEUTX fusion leads to LEUTX re-activation. To our knowledge, this fusion has never been reported previously. Whether the current case represents an example of epithelioid MPNST or a distinct tumor entity remains to be determined.

Paediatric-type diffuse high-grade gliomas in the 5th CNS WHO Classification.

As a relevant element of novelty, the fifth CNS WHO Classification highlights the distinctive pathobiology underlying gliomas arising primarily in children by recognizing for the first time the families of paediatric-type diffuse gliomas, both high-grade and low-grade. This review will focus on the family of paediatric-type diffuse high-grade gliomas, which includes four tumour types: 1) Diffuse midline glioma H3 K27-altered; 2) Diffuse hemispheric glioma H3 G34-mutant; 3) Diffuse paediatric-type high-grade glioma H3-wildtype and IDH-wildtype; and 4) Infant-type hemispheric glioma. The essential and desirable diagnostic criteria as well as the entities entering in the differential will be discussed for each tumour type. A special focus will be given on the issues encountered in the daily practice, especially regarding the diagnosis of the diffuse paediatric-type high-grade glioma H3-wildtype and IDH-wildtype. The advantages and the limits of the multiple molecular tests which may be utilised to define the entities of this tumour family will be evaluated in each diagnostic context.

Mutations in KCNK4 that Affect Gating Cause a Recognizable Neurodevelopmental Syndrome.

Aberrant activation or inhibition of potassium (K+) currents across the plasma membrane of cells has been causally linked to altered neurotransmission, cardiac arrhythmias, endocrine dysfunction, and (more rarely) perturbed developmental processes. The K+ channel subfamily K member 4 (KCNK4), also known as TRAAK (TWIK-related arachidonic acid-stimulated K+ channel), belongs to the mechano-gated ion channels of the TRAAK/TREK subfamily of two-pore-domain (K2P) K+ channels. While K2P channels are well known to contribute to the resting membrane potential and cellular excitability, their involvement in pathophysiological processes remains largely uncharacterized. We report that de novo missense mutations in KCNK4 cause a recognizable syndrome with a distinctive facial gestalt, for which we propose the acronym FHEIG (facial dysmorphism, hypertrichosis, epilepsy, intellectual disability/developmental delay, and gingival overgrowth). Patch-clamp analyses documented a significant gain of function of the identified KCNK4 channel mutants basally and impaired sensitivity to mechanical stimulation and arachidonic acid. Co-expression experiments indicated a dominant behavior of the disease-causing mutations. Molecular dynamics simulations consistently indicated that mutations favor sealing of the lateral intramembrane fenestration that has been proposed to negatively control K+ flow by allowing lipid access to the central cavity of the channel. Overall, our findings illustrate the pleiotropic effect of dysregulated KCNK4 function and provide support to the hypothesis of a gating mechanism based on the lateral fenestrations of K2P channels.

ALK-rearranged histiocytosis: Report of two cases with involvement of the central nervous system.

AIMS: Histiocytoses are a heterogeneous group of localized or disseminated diseases. Clinical presentation and patients' outcome vary greatly, ranging from mild to life-threatening disorders. Rare cases of systemic or localized histiocytosis harboring ALK rearrangement have been reported. METHODS: Two cases of CNS histiocytosis were thoroughly investigated by implementing multiple molecular tests, i.e. FISH, RT-qPCR, NGS analysis. RESULTS: In a 10-month old girl (patient #1), MRI showed two left hemispheric lesions and a right fronto-mesial lesion histologically consisting of a moderately cellular infiltrative proliferation, composed by CD68(PGM1)+/CD163+ spindle cells. ALK 5'/3'-imbalance and a KIF5B(exon 24)-ALK(exon 20) fusion were documented by RT-qPCR and NGS analysis, respectively. A subsequent CT scan showed multiple hepatic and pulmonary lesions. The patient was started on chemotherapy (vinblastine) associated to an ALK-inhibitor (Alectinib) with remarkable response. In a 11-year-old girl (patient #2), MRI showed a right frontal 1.5 cm lesion. Neuropathological examination revealed a histiocytic proliferation composed by medium sized CD68(PGM1)+/HLA-DR+ cells, showing moderate ALK1 positivity. ALK rearrangement and a KIF5B(exon 24)-ALK(exon 20) fusion were demonstrated also in this case. Subsequent CT, 18F-FDG-PET and MRI scans showed the presence of a single right femoral lesion, proved to be a fibrous cortical defect. CONCLUSIONS: In ALK-histiocytoses, CNS involvement may occur as part of a systemic disease or, rarely, as its only primary disease localization, which could remain otherwise asymptomatic. The diagnosis often relies on neuropathological examination of brain biopsy, which may pose a diagnostic challenge due to the variable histopathological features. An integrated histological and molecular approach in such cases is recommended.

CD28.OX40 co-stimulatory combination is associated with long in vivo persistence and high activity of CAR.CD30 T-cells.

The prognosis of many patients with chemotherapy-refractory or multiply relapsed CD30+ non-Hodgkin Lymphoma (NHL) or Hodgkin lymphoma (HL) still remains poor, and novel therapeutic approaches are warranted to address this unmet clinical need. In light of this consideration, we designed and pre-clinically validated a Chimeric Antigen Receptor (CAR) construct characterized by a novel anti-CD30 single-chain variable-fragment cassette, linked to CD3ζ by the signaling domains of two costimulatory molecules, namely either CD28.4-1BB or CD28.OX40. We found that CAR.CD30 T-cells exhibit remarkable cytolytic activity in vitro against HL and NHL cell lines, with sustained proliferation and pro-inflammatory cytokine production, even after multiple and sequential lymphoma cell challenges. CAR.CD30 T-cells also demonstrated anti-lymphoma activity in two in vivo xenograft immune-deficient mouse models of metastatic HL and NHL. We observed that administration of CAR.CD30 T-cells, incorporating the CD28.OX40 costimulatory domains and manufactured in the presence of IL7 and IL15, were associated with the best overall survival in the treated mice, along with the establishment of a long-term immunological memory, able to protect mice from further tumor re-challenge. Our data indicate that, in the context of in vivo systemic metastatic xenograft mouse models, the costimulatory machinery of CD28.OX40 is crucial for improving persistence, in vivo expansion and proliferation of CAR.CD30 T-cells upon tumor encounter. CD28.OX40 costimulatory combination is ultimately responsible for the antitumor efficacy of the approach, paving the way to translate this therapeutic strategy in patients with CD30+ HL and NHL.

Defective kinesin binding of TUBB2A causes progressive spastic ataxia syndrome resembling sacsinopathy.

Microtubules participate in fundamental cellular processes, including chromosomal segregation and cell division, migration and intracellular trafficking. Their proper function is required for correct central nervous system development and operative preservation, and mutations in genes coding tubulins, the constituting units of microtubules, underlie a family of neurodevelopmental and neurodegenerative diseases, collectively known as 'tubulinopathies', characterized by a wide range of neuronal defects resulting from defective proliferation, migration and function. Here, we causally link a previously unreported missense mutation in TUBB2A (c.1249G>A, p.D417N), encoding one of the neuron-specific ß-tubulin isotype II, to a disorder characterized by progressive spastic paraplegia, peripheral sensory-motor polyneuropathy and ataxia. Asp417 is a highly conserved solvent-exposed residue at the site mediating binding of kinesin superfamily motors. Impaired binding to KIF1A, a neuron-specific kinesin required for transport of synaptic vesicle precursors of the disease-associated TUBB2A mutant, was predicted by structural analyses and confirmed experimentally in vitro. We show that overexpression of TUBB2AD417N disrupts the mitotic spindle bipolarity and morphology and affects the M phase entry and length. Differently from the TUBB2AN247K and TUBB2AA248V, two mutants previously identified to affect neurodevelopment, TUBB2AD417N retains the ability to assemble into microtubules. Consistent with the differential clinical and structural impact, TUBB2AA248V does not drastically affect TUBB2A binding to KIF1A, nor mitotic spindle bipolarity. Overall, our data demonstrate a pathogenic role of the p.D417N substitution that is different from previously reported TUBB2A mutations and expand the phenotypic spectrum associated with mutations in this gene.

Skeletal abnormalities are common features in Aymé-Gripp syndrome.

Aymé-Gripp syndrome (AYGRPS) is a recognizable condition caused by a restricted spectrum of dominantly acting missense mutations affecting the transcription factor MAF. Major clinical features of AYGRPS include congenital cataracts, sensorineural hearing loss, intellectual disability, and a distinctive flat facial appearance. Skeletal abnormalities have also been observed in affected individuals, even though these features have not been assessed systematically. Expanding the series with four additional patients, here we provide a more accurate delineation of the molecular aspects and clinical phenotype, particularly focusing on the skeletal features characterizing this disorder. Apart from previously reported malar flattening and joint limitations, we document that carpal/tarsal and long bone defects, and hip dysplasia occur in affected subjects more frequently than formerly appreciated.

Co-occurrence of mutations in KIF7 and KIAA0556 in Joubert syndrome with ocular coloboma, pituitary malformation and growth hormone deficiency: a case report and literature review.

BACKGROUND: Joubert syndrome is a recessive neurodevelopmental disorder characterized by clinical and genetic heterogeneity. Clinical hallmarks include hypotonia, ataxia, facial dysmorphism, abnormal eye movement, irregular breathing pattern cognitive impairment and, the molar tooth sign is the pathognomonic midbrain-hindbrain malformation on magnetic resonance imaging. The disorder is predominantly caused by biallelic mutations in more than 30 genes encoding proteins with a pivotal role in morphology and function of the primary cilium. Oligogenic inheritance or occurrence of genetic modifiers has been suggested to contribute to the variability of the clinical phenotype. We report on a family with peculiar clinical spectrum Joubert syndrome molecularly and clinically dissecting a complex phenotype, in which hypogonadism, pituitary malformation and growth hormone deficiency occur as major features. CASE PRESENTATION: A 7 year-old male was enrolled in a dedicated "Undiagnosed Patients Program" for a peculiar form of Joubert syndrome complicated by iris and retinochoroidal coloboma, hypogonadism pituitary malformation, and growth hormone deficiency. The molecular basis of the complex phenotype was investigated by whole exome sequencing. The concomitant occurrence of homozygosity for mutations in KIF7 and KIAA0556 was identified, and the assessment of major clinical features associated with mutations in these two genes provided evidence that these two independent events represent the cause underlying the complexity of the present clinical phenotype. CONCLUSION: Beside the clinical variability of Joubert syndrome, co-occurrence of mutations in ciliopathy-associated genes may contribute to increase the clinical complexity of the trait.

NBAS pathogenic variants: Defining the associated clinical and facial phenotype and genotype-phenotype correlations.

The pathogenic variants in the neuroblastoma-amplified sequence (NBAS) are associated with a clinical spectrum involving the hepatic, skeletal, ocular, and immune systems. Here, we report on two unrelated subjects with a complex phenotype solved by whole-exome sequencing, who shared a synonymous change in NBAS that was documented to affect the transcript processing and co-occurring with a truncating change. Starting from these two cases, we systematically assessed the clinical information available for all subjects with biallelic NBAS pathogenic variants (73 cases in total). We revealed a recognizable facial profile (hypotelorism, thin lips, pointed chin, and "progeroid" appearance) determined by using DeepGestalt facial recognition technology, and we provide evidence for the occurrence of genotype-phenotype correlations. Notably, severe hepatic involvement was associated with variants affecting the NBAS-Nter and Sec39 domains, whereas milder liver involvement and immunodeficiency were generally associated with variants located at the N-terminus and C-terminus of the protein. Remarkably, no patient was reported to carry two nonsense variants, suggesting lethality of complete NBAS loss-of-function.

TBCE Mutations Cause Early-Onset Progressive Encephalopathy with Distal Spinal Muscular Atrophy.

Tubulinopathies constitute a family of neurodevelopmental/neurodegenerative disorders caused by mutations in several genes encoding tubulin isoforms. Loss-of-function mutations in TBCE, encoding one of the five tubulin-specific chaperones involved in tubulin folding and polymerization, cause two rare neurodevelopmental syndromes, hypoparathyroidism-retardation-dysmorphism and Kenny-Caffey syndrome. Although a missense mutation in Tbce has been associated with progressive distal motor neuronopathy in the pmn/pmn mice, no similar degenerative phenotype has been recognized in humans. We report on the identification of an early-onset and progressive neurodegenerative encephalopathy with distal spinal muscular atrophy resembling the phenotype of pmn/pmn mice and caused by biallelic TBCE mutations, with the c.464T>A (p.Ile155Asn) change occurring at the heterozygous/homozygous state in six affected subjects from four unrelated families originated from the same geographical area in Southern Italy. Western blot analysis of patient fibroblasts documented a reduced amount of TBCE, suggestive of rapid degradation of the mutant protein, similarly to what was observed in pmn/pmn fibroblasts. The impact of TBCE mutations on microtubule polymerization was determined using biochemical fractionation and analyzing the nucleation and growth of microtubules at the centrosome and extracentrosomal sites after treatment with nocodazole. Primary fibroblasts obtained from affected subjects displayed a reduced level of polymerized α-tubulin, similarly to tail fibroblasts of pmn/pmn mice. Moreover, markedly delayed microtubule re-polymerization and abnormal mitotic spindles with disorganized microtubule arrangement were also documented. Although loss of function of TBCE has been documented to impact multiple developmental processes, the present findings provide evidence that hypomorphic TBCE mutations primarily drive neurodegeneration.